JP5419279B2 - Improved stem cell culture - Google Patents

Description

Detailed Description of the Invention

This application claims the benefit of US Provisional Patent Application No. 60 / 880,747 on January 17, 2007, the description of which is incorporated herein by reference in its entirety. And

Embryonic stem (ES) cells are pluripotent cells that are both pluripotent in nature, capable of both cell culture growth and differentiation into various lineage-restricted cell populations (Odorico et al., Stem Cells 19: 193-204 (2001)). Because of these properties, ES cells, including human ES cells, can be very specific cell types that perform various functions.
In general, human ES cells are highly uniform, have the ability to self-renew, and have the ability to differentiate into any functional cell in the body. Self-renewal leads to a long-term proliferative capacity with the possibility of unlimited growth in cell culture under appropriate conditions. Furthermore, when human ES cells are differentiated so as not to be site-specific, a heterogeneous population of cells expressing markers of different tissue types is obtained (WO 01/51616; Shamblott et al., Proc. Natl. Acad. Sci. USA 98: 113 (2001)). These features make human ES cells a unique homogeneous starting population for the production of cells with therapeutic utility.
Human ES cells can be used to make various differentiated cell types for scientific and commercial research purposes. Currently, many types of differentiated human cells are not readily available and cannot grow to a significant number in vitro culture. However, human ES cells can grow indefinitely in culture and can differentiate into many, but not all, differentiated cell types of the human body. Thus, culture techniques have been developed to induce human ES cells to differentiate into many specific cell types in the human body. The availability of human ES cells has opened up the possibility that many differentiated human cells will be available in significant numbers for scientific and commercial research.
One difficulty in working with human ES cells is to develop standard culture conditions for human ES cells without using products such as animal products or serum, which tend to vary between batches . Thus, this technique requires that the culture conditions of human ES cell culture be as limited as possible.
In order to solve this requirement, a set of culture conditions has recently been described that allow long-term culture of undifferentiated human ES cells under defined conditions. The description of Ludwig et al., Nat. Methods 3: 637-646 (2006) is intended to be included as generally indicated herein. Ludwig et al. Described a medium for culturing human ES cells, referred to therein as TeSR ^™ medium, in which each component of the medium was fully disclosed and confirmed. Therefore, TeSR ^™ is a completely limited and sufficient medium for human ES cell culture. TeSR ^™ has also been shown to be effective for use in the induction of new human ES cell lines, a more difficult limitation than culturing undifferentiated human ES cells.
When cells grow in an environment where they are in direct contact with other cells or with physical structures in the environment, human ES cells preferentially remain undifferentiated. In other cellular environments, human ES cells begin to differentiate and become infinitely proliferating.
This is important in the method of cloning ES cell cultures. “Cloning” as used herein means a method of initiating ES cell culture from a starting culture, ideally from a single ES cell or at least from very few ES cells. Culture conditions that allow clonal culture of undifferentiated ES cells can be all the harshest conditions required for normal ES cell culture and expansion.

In a first aspect, the present invention is a method for cloning a pluripotent stem cell culture, comprising preparing a culture of pluripotent stem cells in a culture in a limited culture medium in a culture vessel; Selecting a single cell from the stem cells in the product; and starting a new culture of the stem cells in the culture medium in a culture vessel having the single cells, wherein the culture medium increases the cloning efficiency of the stem cell culture Summarized as the above method, which is a small molecule that contains the drug of choice and that increases the cloning efficiency of stem cell culture. By "defined culture medium" or "defined medium" is meant that the medium is known for the amount of all components. Typically, serum normally added to the culture medium for cell culture is, for example, a known amount of serum components such as albumin, insulin, transferrin, and possibly certain growth factors (i.e. basic fibroblasts). Growth factor, transforming growth factor or platelet derived growth factor).
In a second embodiment, the present invention is selected to increase the cloning efficiency of a pluripotent stem cell culture comprising a culture vessel; a limited culture medium contained within the vessel; a stem cell growing in the culture medium; and a stem cell culture Summarized as pluripotent stem cell culture containing drug in culture medium.
In some embodiments of any aspect, the agent is a kinase inhibitor. In other embodiments of any aspect, the agent is a protein kinase A inhibitor, protein kinase C inhibitor, protein kinase G inhibitor or Rho-related kinase inhibitor. In still other embodiments of either aspect, the agent is H-7, isoH-7, H-8, H-9, H-89, HA-100, HA-1004, HA-1077, H- Selected from 1152 or Y-27632.

  An object of the present invention is to improve techniques for culturing pluripotent stem cells, including increasing the cloning efficiency of culture conditions. A pluripotent stem cell means an ES cell or an induced pluripotent stem (iPS) cell. iPS cells may vary with respect to the origin of differentiated somatic cells, may vary with respect to a specific set of efficacy determinants, and may vary with respect to the culture conditions used to isolate Good reprogrammed differentiated somatic cells, but nevertheless substantially similar to the origin of the respective differentiated somatic cells and higher potency cells, such as ES cells Show properties.

An advantage of the present invention is that the cloning efficiency of stem cell culture conditions can be improved by adding one of a group of small molecules to the stem cell culture, so that the small molecule is economical and effective.
These and other features, aspects and advantages of the present invention will be better understood from the following description. The description of the preferred embodiments is not intended to limit the invention, but encompasses all modifications, equivalents and alternatives. Therefore, reference should be made to the claims herein for interpreting the scope of the invention.

As described above, the problem of growing pluripotent cells such as ES cells in culture is that the cloning efficiency is low. As used herein, “cloning efficiency” refers to the number of cells individualized with trypsin divided by the number of individual cells applied to a well of a culture dish. In our previous experience of growing human ES cells in conditions free of animal material limited on a matrix (e.g. Matrigel®), the cloning efficiency was very low (i.e., 0.1 %Less than). This is in contrast to previous experience with culture-based culture systems conditioned by exposure to fibroblasts, where cloning efficiency remained low (i.e., less than 2%), but ES cells It was high enough to initiate clonal colonies. As disclosed herein, when a small molecule is added to a culture medium in which human ES cells grow, ES cell culture can be clonally cultured in a very difficult way without the addition of small molecules. Several small molecules are exemplified herein for this purpose.
For clarity, there is a difference between “passaging” human ES cells and initiating clonal colonies. In a typical experiment of ES cell culture, if the culture vessel is the largest, the colony is divided into aggregates and then placed in a new culture vessel. These aggregates typically contain 100-1,000 cells and readily initiate in-culture growth. In contrast, initiating clonal colonies requires growing human ES cell colonies from individual single ES cells.
Small molecules identified herein that act as agents that increase the cloning efficiency of ES cells were identified by a small molecule screen for this purpose. The mechanism by which these small molecules enhanced the cloning efficiency of human ES cell culture is unclear. Perhaps a small molecule interacts with a signaling kinase to change the signaling pathway in the cell, but the identity of the interaction and pathway is currently unknown. Regardless of the mechanism of action, small molecules effectively increased the cloning efficiency of ES cell cultures, even when the culture conditions were different.

Screens were performed using high-throughput fluorescence analysis and screened for the ability to enhance cloning efficiency for small molecules. A non-fluorescent soluble substrate (6,8-difluoro-4-methylumbelliferyl phosphate, DiFMUP) was converted to a fluorescent product by an enzyme called alkaline phosphatase (ALP) specific for undifferentiated human ES cells.
Increased fluorescence from DiFMUP hydrolyzed by ALP in wells formed after 12 days of seeding 500 individualized human ES cells in 96-well plates with more than 2,000 cells allowing colony formation Any well containing the compound was shown. Proliferated cells lost ALP activity upon differentiation and did not increase in fluorescence. Thus, this analysis examined both clonal expansion and maintenance of undifferentiated state.
The method described herein was used to identify a set of small molecules that increase ES cell cloning efficiency. One preferred small molecule is (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl] homopiperazine dihydrochloride (informal name: H-1152). Another preferred small molecule is 1- (5-isoquinolinesulfonyl) piperazine hydrochloride (unofficial name: HA-100). Although both appear to be equally effective in facilitating clonal growth, H-1152 can be used at a working concentration 10 times lower than HA-100. Other related small molecules that were effective included: 1- (5-isoquinolinesulfonyl) -2-methylpiperazine (unofficial name: H-7), 1- (5-isoquinolinesulfonyl) ) -3-Methylpiperazine (Informal name: IsoH-7), N-2- (Methylamino) ethyl-5-isoquinolinesulfonamide dihydrochloride (Informal name: H-8), N- (2- Aminoethyl) -5-isoquinolinesulfonamide dihydrochloride (informal name: H-9), N- [2-p-bromocinnamylamino) ethyl] -5-isoquinolinesulfonamide dihydrochloride (informal name: H-89), N- (2-guanidinoethyl) -5-isoquinolinesulfonamide hydrochloride (unofficial name: HA-1004), 1- (5-isoquinolinesulfonyl) homopiperazine dihydrochloride (unofficial name: HA -1077), (S)-(+)-2-methyl-4-glycyl-1- (4-methylisoquinolinyl-5-sulfonyl) homopiperidine dihydrochloride (unofficial name: glycyl H-1152) , (+)-(R) -trans-4- (1-amino Noethyl) -N- (4-pyridyl) cyclohexanecarboxamide dihydrochloride (unofficial name: Y-27632). Each small molecule supported a cloning efficiency> 1% in a defined medium on a Matrigel®-coated culture dish, eg, TeSR ^™ 1 medium. All components of TeSR ^™ 1 medium and methods of use are described above in Ludwig et al. Ludwig et al.

The effects coordinated by these small molecules were not limited to the use of TeSR ^™ 1 medium. These small molecules also increased the cloning efficiency of ES cell cultures grown on conditioned medium, a medium exposed to fibroblasts. Thus, these small molecules are thought to increase the cloning efficiency of any ES cell culture medium in which ES cells can be effectively propagated.
One type of small molecule that is effective in increasing the cloning efficiency of ES cell culture media includes protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), and Rho-related kinase (ROCK) An inhibitor of kinase enzymes.
ROCK is of particular interest herein. ROCK is a serine / threonine kinase that serves as a target protein for Rho (there are three isoforms--RhoA, RhoB and RhoC). These kinases were first identified as RhoA-induced stress fiber formation and focal adhesion chemical mediators. Two ROCK isoforms--ROCK1 (p160ROCK, also referred to as ROKβ) and ROCK2 (ROKα)-are from a coiled-coil domain containing an N-terminal kinase domain followed by a Rho-binding domain and a pleckstrin-homology domain (pH). Composed. Both ROCKs are cytoskeletal regulators that mediate RhoA action on stress fiber formation, smooth muscle contraction, cell adhesion, membrane roughing and cell motility. ROCK exhibits biological activity by targeting downstream molecules such as myosin light chain (MLC), MLC phosphatase (MLCP), phosphatase and tensin analogs (PTEN).
An exemplary ROCK inhibitor is Y-27632, which selectively targets ROCK1 (also inhibits ROCK2) and inhibits TNF-α and IL-1β. It is cell permeable and inhibits ROCK1 / ROCK2 (IC ₅₀ = 800 nM) by competing with ATP. The description of Ishizaki T, et al., Mol. Pharmacol. 57: 976-983 (2000) is intended to be included as generally indicated herein. Examples of other ROCK inhibitors include H-1152, Y-30141, Wf-536, HA-1077, hydroxyl HA-1077, GSK269962A and SB-772077-B. Doe C, et al., J. Pharmacol. Exp. Ther. 32: 89-98 (2007); Ishizaki et al .; Nakajima M, et al., Cancer Chemother. Pharmacol. 52: 319-324 (2003) The descriptions of Sasaki Y, et al., Pharmacol. Ther. 93: 225-232 (2002) are each intended to be included as generally indicated herein.
Small molecules identified herein have at least pyridine as a common structural element. Thus, other small molecules useful herein include, for example, N- (4-pyridyl) -N ′-(2,4,6-trichlorophenyl) urea, 3- (4-pyridyl)- 1H-indole, (R)-(+)-trans-N- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide.
The data shown below and described above demonstrated that one of the small molecules generally increases the cloning efficiency of human ES cell culture media. In general, one type of effective small molecule can be summarized as follows:

Wherein R ₁ and R ₂ can be bonded via an aromatic group, R ₁ is hydrogen or alkyl, R ₂ is hydrogen or alkyl, and R ₁ and R ₂ are cyclic Unless it is part of an aromatic, R ₁ is not the same as R ₂ and R ₃ and R ₄ are bonded to any atom of isoquinoline and are hydrogen or alkyl).

Example 1: Identification of small molecules that increase ES cell cloning efficiency
Methods and Materials For small molecules that are effective in allowing cloning of ES cell colonies in limited media, the desired activity that allowed cloning was identified in a small molecule screen.
The screen used a small molecule library made up of compounds confirmed by known biological activities purchased from Sigma-Aldrich (St. Louis, MO). The library type was dissolved in DMSO to a concentration of 1 mM. Unless otherwise indicated, all other chemicals used were purchased from Sigma. H1 human ES cells (WiCell Research Institute; Madison, Wis.) Were grown in limited TeSR ^™ 1 medium prepared as described above in Ludwig et al.
The analysis was performed in a 96-well microplate using a Beckman Biomek® FX laboratory workstation and a Perkin Elmer VictorIII® plate reader at the Keck-UWCCC small molecule screening facility (University of Wisconsin-Madison, Madison, WI). Nunclon / Nunc; Roskilde, Denmark). Microplates were coated with Matrigel® (# 354230, reduced growth factor; BD Bioscience, San Jose, Calif.) In DMEM / F12 (Invitrogen; Carlsbad, Calif.) At 0.3 mg / ml for 1 hour at room temperature. .
H1 human ES cells were grown in 6-well Nunclon® dishes on day 5 and then treated with 1 ml trypsin / EDTA (Invitrogen) containing 1% chicken serum for 5 minutes at 37 ° C. Trypsin was quenched with 20% fetal calf serum in DMEM / F12 and cells were collected by centrifugation at 200 RCF for 1.5 minutes. After aspirating the medium, the cells were resuspended in TeSR ^™ 1 medium to a density of 5,000 cells / ml. 100 μL of this cell suspension was dispensed into each well, 1 μL of 10 mM inhibitor DMSO solution was added and mixed by pipetting to obtain a final inhibitor concentration of 10 μM. Plates were incubated at 37 ° C. for 2 days with 5% CO ₂ . The medium was then replaced with TeSR ^™ 1 medium without cloning molecules. The plate was further cultured for 8 days and the medium was changed every other day.
In order to detect colony formation, we used ALP analysis (EnzChek®; Invitrogen) by fluorescence analysis. In this analysis, soluble non-fluorescent substrate (DiFMUP) is converted to a fluorescent product by ALP. The plate was washed with 50 mM TrisHCl (pH 7.5) and treated with 100 μL of 38 μM DiFMUP solution for 1 hour. In wells with colonies, an increase in fluorescence was shown for any well containing compounds that lead to colony formation. Proliferated but differentiated cells did not produce an increase in fluorescence as these cells lack ALP activity. The plate was read by a multiplate reader (excitation 355 nm, emission 455 nm with 0.1 ms emission interval).
Compounds that showed activity in the screen were tested in 6-well plates at various concentrations in 1% DMSO. For these analyses, 10,000 cells / well were plated on Matrigel® coated plates and then treated with the small molecules every other day for a total of 4 days. To evaluate the effect of the small molecules tested on cloning efficiency, we counted colonies by hand after 10 days.
We also tested the effects of these small molecules on human ES cell cloning on other media. We tested the effect on conditioned medium (CM) by replacing TeSR ^™ 1 medium with CM or the same treatment. The cloning efficiency of the medium increased with the addition of HA-100. In addition, we performed the same test with unconditioned medium supplemented with 100 ng / μl bFGF. Furthermore, the addition of HA-100 increased the cloning efficiency compared to similar control cultures lacking HA-100.

Results The results of the small molecule screen were in relative fluorescence units. The typical performance of the fluorescence analysis gave a mean value of 17,868, a standard deviation of 5,104 and a standard error of 197. Over 200,000 records were obtained with typical success (meaning that colonies grew). All potential successes were confirmed by visual inspection of sufficient samples. Obvious colonies of approximately 1 mm diameter were observed in the wells as true positives. We got 4 successes from 4,500 individual samples. Success was HA-100, latosterol (cholesterol precursor), obacnol (limonoid) and quipazine dimaleate (serotonin 2A agonist). From these four compounds, only HA-100 could be confirmed as active in increasing the cloning efficiency of human ES cells cultured in a 6-well assay.
We determined the cloning efficiency (CE; number of colony forming units (CFU) per number of applied single cells, treated with trypsin / EDTA). HA-100 is the first molecule identified in the small molecule screen, after which cloning efficiency was also tested for other structurally similar small molecules. The following compounds show similar activity within a range of concentrations, and IC ₅₀ values for three kinases (e.g., PKA, PKC, PKG) by small molecules are shown in Table 1 below:
HA-100 (Sigma) having the following structure:

  H-7 (Sigma) having the following structure:

  Iso H-7 (Sigma) having the following structure:

  H-89 (Sigma) having the following structure:

  HA-1004 (Sigma) having the following structure:

table 1

In addition, all small molecules were tested in conditioned medium (CM) and showed a cloning efficiency of up to 30% CM with small molecules compared to CM alone.
We also tested colonies obtained from HA-100 treatment in which an intracellular marker for Oct4, a pluripotent ES cell, was present. Immunofluorescence showed clear evidence of maintenance of Oct4 expression in cells treated in cloned colonies, implying an undifferentiated state. This indicates that human ES cells are individualized with trypsin and can be cultured in the presence of HA-100 without loss of pluripotency.
Subsequently, other related small molecules were also tested for their ability to increase the cloning efficiency of human ES cells. HA-1077 (Upstate Biotechnologies, Lake Placid, NY) having the following structure increased cloning efficiency:

  Two other related small molecules also showed similar effects. The first small molecule was H-8 (Biomol; Plymouth Meeting, PA) having the following structure:

  The second small molecule was H-9 (Biomol) with the following structure:

  However, one small molecule did not increase the cloning efficiency of human ES cells. This small molecule has 1- [N, O-bis- (5-isoquinolinesulfonyl) -N-methyl-L-tyrosyl] -4-phenylpiperazine (informal name: KN-62 or KN62) having the structure Met:

Example 2: ROCK inhibitor increases ES cell cloning efficiency
Methods and Materials The experiment described in Example 1 was repeated with H-1152 (EMD Chemicals, Inc./Calbiochem; San Diego, CA) and Y-27632 (EMD Chemicals, Inc./Calbiochem). As a summary, H-1152 or Y-27632 was dissolved in DMSO to a concentration of 20 mM. H1 human ES cells were grown at a density of 100,000 cells / ml on Matrigel®-coated plates in TeSR ^™ 1 medium. H-1152 and Y-27632 were added to wells on their own plates at final concentrations of 20 μM, 15 μM, 10 μM or 5 μM / DMSO. DMSO and HA-100 (10 μM) were used as negative and positive controls, respectively. Plates were processed every other day for a total of 4 days. Colony formation was detected in the ALP analysis.
In a second experiment, H-1152 and Y-27632 were added to wells on their own plates at final concentrations of 5 μM, 500 nM, 50 nM or 5 nM / DMSO. DMSO and HA-100 (10 μM) were used as negative and positive controls, respectively. Plates were processed as described above.
result
500 nM H-1152 and 5 μM Y-27632 showed most colony formation in ALP analysis. Examine the plate visually for H-1152 at concentrations greater than 500 nM, and for Y-27632 at concentrations greater than 5 μM, may be the same or better than HA-100 (10 μM). It was seen.
The structure of H-1152 is as follows:

  The structure of Y-27632 is as follows:

  Although the foregoing invention has been described in detail by way of illustration and example for clarity of understanding, certain adaptations of the invention are part of the normal optimization for those skilled in the art and are It will be understood that the invention can be practiced without departing from the scope of the appended claims.

Claims (10)

A method for generating a clonal culture of human pluripotent stem cells comprising:
Preparing a culture of human pluripotent stem cells in a culture in a limited culture medium containing basic fibroblast growth factor (bFGF) in a culture vessel;
Selecting a single human pluripotent stem cell from the stem cells in culture ;
Using a single human pluripotent stem cell, starting a new culture of human pluripotent stem cells in a culture vessel,
The culture container contains a matrix that covers the surface of the container, and a bFGF-containing limited culture medium,
bFGF-containing limited culture medium contains drug,
The drug is selected to increase the cloning efficiency of human pluripotent stem cell cultures compared to bFGF-containing limited culture medium that does not contain the drug , and the following:

A small molecule represented by a formula selected from the group consisting of :
Culturing a single human pluripotent stem cell to produce a clonal culture of human pluripotent stem cells .   The method of claim 1, wherein the agent is a kinase inhibitor. The method of claim 1, wherein the agent is a Rho-related kinase (ROCK) inhibitor. 2. The method according to claim 1, wherein the human pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell. bFGF-containing limited culture medium, we support the maintenance and growth of pluripotent stem cells undifferentiated no Feeder cells or serum, the method according to claim 1. A method for generating a clonal culture of human pluripotent stem cells comprising:
Preparing a culture of in-culture human pluripotent stem cells in a bFGF-containing limited culture medium in a culture vessel;
Selecting a single human pluripotent stem cell from the stem cells in culture ;
Using single pieces of human pluripotent stem cells, comprising the steps of starting a new culture of human pluripotent stem cells in culture vessel,
The culture container contains a bFGF-containing limited culture medium,
bFGF-containing limited culture medium contains drug,
The agent is selected to increase the cloning efficiency of human pluripotent stem cell cultures compared to a pluripotent stem cell culture medium that does not contain the agent , and the following:

Said step represented by a formula selected from the group consisting of : and
Culturing the single pieces of human pluripotent stem cells, comprising the step <br/> of generating clonal culture of human pluripotent stem cells, the how. The method according to claim 6 , wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. Culture vessels;
BFGF-containing limited culture medium , contained in a container;
human pluripotent stem cells growing in a bFGF-containing limited culture medium; and
A bFGF-containing limited culture medium containing drug agent,
The drug is selected to increase the cloning efficiency of human pluripotent stem cell culture compared to bFGF-containing limited culture medium that does not contain the drug ,
The drug is :

Represented by a formula selected from the group consisting of a kinase inhibitor, including the culture medium, human pluripotent stem cell culture. Stem cells are embryonic stem cells or induced pluripotent stem cells, human pluripotent stem cell culture according to claim 8. A limited culture locations for culturing human pluripotent stem cells, comprising an agent selected to increase the cloning efficiency of the human pluripotent stem cell culture, drug, the following:

The culture medium represented by a formula selected from the group consisting of: